Bisphosphonic acids are a group of compounds having a P—C—P skeleton, and show high bone tissue penetration and high affinity for bone. In addition, when the first generation bisphosphonic acids such as etidronic acid, clodronic acid and the like are selectively incorporated into monocytic cells such as osteoclasts and the like by liquid-phase endocytosis, they are metabolically converted to ATP analogs, act antagonistically to ATP receptors and show cytotoxicity. Thus, the first generation bisphosphonic acids suppress bone resorption by inducing cell death in osteoclasts. Utilizing such property, bisphosphonic acids are applied to various bone-related diseases. To be specific, they are used as prophylactic or therapeutic drugs for diseases relating to the fragility of bone and calcium concentration variation such as osteoporosis, osteitis deformans, osteogenesis imperfecta and hypercalcemia in malignant tumor. In addition, bisphosphonic acids belonging to the second generation such as pamidronic acid, alendronic acid, ibandronic acid and the like, and bisphosphonic acids belonging to the third generation such as risedronic acid, zoledronic acid and the like contain a nitrogen atom in the side chain, and are called nitrogen-containing bisphosphonic acids. When these bisphosphonic acids are selectively incorporated into monocytic cells such as osteoclast and the like, they specifically inhibit farnesyl diphosphate synthase and show cytotoxicity. Utilizing the properties thereof, various nitrogen-containing bisphosphonic acids have been used as improving-drugs for osteoporosis and hypercalcemia in malignant tumor. Recently, moreover, it has been reported that the disease-free survival is preferentially extended when zoledronic acid is used as an adjuvant therapy drug in the endocrine therapy and chemotherapy of premenopausal estrogen sensitive early breast cancer cases and multiple myeloma (non-patent documents 1, 2). This is considered to be because nitrogen-containing bisphosphonic acid has direct cytotoxicity and/or indirect cytotoxicity via activation of immunocytes on tumor cells and shows an antitumor effect.
For example, a part of etidronic acid or clodronic acid administered to a living body enters into the cell by a fluid phase endocytosis action, is transferred to nucleoside monophosphate, and converted to a nucleoside triphosphate analog compound. A metabolite thereof is shown to antagonistically inhibit biological enzyme reaction utilizing high energy phosphate bond of nucleoside triphosphate. When the incorporating cell is osteoclast, bone resorption is suppressed, and the concentration of calcium in the plasma decreases. In the case of tumor cells, the tumor cells are injured and a direct antitumor effect is expected.
The second generation and third generation nitrogen-containing bisphosphonic acids transferred into the cell have been shown to inhibit farnesyl diphosphate synthase involved in the biosynthesis pathway of isoprenoidal metabolites such as cholesterol and the like. Such enzyme catalyzes a reaction to synthesize geranyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, and a reaction to synthesize farnesyl diphosphate from isopentenyl diphosphate and geranyl diphosphate. Therefore, inhibition of farnesyl diphosphate synthase is considered to shut off the metabolic pathway located downstream of geranyl diphosphate, as well as cause accumulation of isopentenyl diphosphate to be an enzyme substrate. When the biosynthesis pathway located downstream of geranyl diphosphate is shut off, isoprenoidal compounds such as cholesterol, liposoluble vitamins, bile acid, lipoprotein and the like are not biosynthesized, and the proliferation of tumor cells is considered to be suppressed.
Generally, the isopropenyl group of farnesyl diphosphate and geranylgeranyl diphosphate biosynthesized by farnesyl diphosphate synthase is transferred to, what is called, small G proteins such as Ras, Rho, Rap, Rab, Rac and the like. The small G protein having the transferred isopropenyl group is translocated to a cellular membrane, which is an inherent action site of small G protein, since the isopropenyl group functions as a cellular membrane anchor, and exhibits important physiological functions such as cell proliferation, adhesion and the like. However, when nitrogen-containing bisphosphonic acid such as zoledronic acid and the like inhibits farnesyl diphosphate synthase, transfer of the isopropenyl group is inhibited, translocation to the membrane of small G protein is prevented, and, as a result, tumor cell proliferation is inhibited. This is one of the mechanisms that explain direct antitumor effects shown by nitrogen-containing bisphosphonic acid.
When farnesyl diphosphate synthase is further inhibited, the intracellular concentration of isopentenyl diphosphate as a substrate thereof increases. The increase in the intracellular concentration of isopentenyl diphosphate is detected by a butyrophilin 3A1 transmembrane type protein, and the change thereof is recognized by γδ T cells having a Vγ2Vδ2 T cell receptor (non-patent documents 3, 4). As a result, the γδ T cells are degranulated to release perforin and granzyme B, which induces apoptosis of tumor cells and virus infected cells. It is shown that nitrogen-containing bisphosphonic acid indirectly and efficiently damage tumor cells and virus-infected cells via activation of immunocyte.
The direct and indirect cytotoxicity by the nitrogen-containing bisphosphonic acids as mentioned above depends on the degree of incorporation into the cells to be injured, and the degree of inhibition of farnesyl diphosphate synthase. However, since bisphosphonic acids clinically applicable at present have all been synthesized for the purpose of improving bone-related disease, synthesis and screening of the compounds was performed using the affinity to bone, which is the action site of osteoclast, and cytotoxicity to osteoclast as indices. However, in the development of medicaments against tumor and virus infection, high bone penetration is conversely a factor that decreases reachability to tumor cells and virus infected cells.
Therefore, when direct improvement of cytotoxicity is desired, a decrease in the bone penetration needs to be one goal. On the other hand, when improvement of activation of γδ T cell as an immunity effector is desired, it is necessary to develop drugs by using, as indices, uptake into monocyte cells to be antigen presenting cells and γδ T cells activation potency. Thus, for compound screening without using suppression of bone resorption as an index, systematic synthesis of bisphosphonic acid having a basic skeleton different from that of conventional bisphosphonic acid is necessary.
About 30% of the low molecular medicaments currently on the market have fluorine in the basic skeleton. The reason for the superiority of medicaments due to the presence of a fluorine atom has not been completely elucidated. Until now, however, in the developmental stage of bisphosphonic acid, the fluorine-containing bisphosphonic acid is only one in which the hydroxyl group bonded to C in the P—C—P skeleton of risedronic acid is substituted by fluorine. This is because introduction of a fluorine atom is synthetically difficult in bisphosphonic acids. Therefore, it is an important research progress in the development of a medicament of bisphosphonic acid to explore a synthetic pathway of a series of fluorine-containing bisphosphonate derivatives, synthesize them systematically and study their physiological activities.